Over 50% of human cancers contain mutations in the tumor suppressor gene coding for p53 protein. p53 is activated by a cellular pathway that responds to DNA damage caused by X-rays, u.v. light, mutagens and oxidation, and leads to a block in the cell cycle to allow time for repair, or apoptosis. Relatively little is known about the molecular signals which alert p53 to the presence of DNA damage. Recently, using electron microscopy (EM) and gel shift analysis it was shown that p53 binds to DNA at sites containing bulged bases (IDL mismatches) for example a bulge of 3 cytosines or 4 CA repeats. Further, the p53 complexes formed at the lesions had a half life of >2 hours while complexes formed with DNA containing only free ends were unstable. These stable (lesion) complexes were much stronger than those formed at the transcriptional MCK p53 response element. This led to the working hypothesis that p53 is able to directly recognize a variety of lesions on DNA and that the formation of stable complexes at the lesions signals the presence of damage to the cell -- thus activating certain pathways and possibly helping to recruit the repair proteins. These findings open the door to detailed studies focused on determining the full spectrum of DNA damage recognition by p53. Here DNA molecules containing specific lesions at their center will be constructed. Lesions will include extra base bulges, single base mismatches, and damage due to u.v. irradiation, mutagens, and oxidative insult. Using human p53, each DNA will be subjected to quantitative binding analysis using EM, filter binding, and gel shift methods. Competitive gel shift and filter binding experiments will provide kw values and a scale of affinities for the different lesions -- which will be compared to several p53 response elements. Experiments with the human postreplication mismatch repair proteins and p53 will probe questions of cocomplex formation or competition for lesion binding. Studies of p53 mutants will examine the cooperation between the C terminus and central domain of p53 and help explain why these mutations in p53 lead to cancer. EM will provide a description of the oligomeric state of p53 at the lesion, and a measure of p53 binding at the damage as contrasted to DNA ends or elsewhere. EM will reveal whether the stable complexes formed at the lesion will loop DNA to facilitate distant interactions at p53 response elements. The relevance of this work to cancer lies in the central role that p53 plays in tumor development and the possibility that by understanding how it functions, better anti-tumor therapies can be designed.